The Hsp90 molecular chaperone has emerged as a promising target for the treatment of cancer. While most current therapies are directed at disrupting a single molecular function, Hsp90 is distinctive in its regulation of multiple oncogenic pathways. There are more than 100 client proteins that depend upon Hsp90 for their folding and conformational maintenance, many of which contribute to cancer cell proliferation. Hsp90 inhibition is similar to a combinatorial attack, in which multiple signaling pathways that contribute to malignancy are simultaneously disrupted. See generally Blagg, U.S. Published Patent Application Nos. 2006/0199776 and 2007/0270452, which are incorporated by reference in their entirety.
Novobiocin, a member of the coumermycin family of antibiotics, was isolated from streptomyces and shown to manifest potent activity against Gram-positive bacteria. Novobiocin elicits antimicrobial activity through binding the ATP-binding pocket of DNA gyrase and prohibiting ATP-hydrolysis. Co-crystal structures of GyrB, the B subunit of DNA gyrase, bound to novobiocin and ADP revealed both molecules bind in a bent conformation, which is in contrast to most ATP-binding proteins that bind ADP in an extended conformation. Realizing Hsp90 binds ADP in a similar conformation as that observed for Gyrase, Neckers and co-workers hypothesized and subsequently demonstrated that novobiocin binds Hsp90 and exhibits anti-tumor activity (about 700 μM) against breast cancer cells. Upon Western blot analyses of SKBr3 cells, Neckers and co-workers demonstrated that novobiocin induces degradation of Hsp90-dependent clients in a concentration-dependent manner, suggesting Hsp90 is the biological target. In contrast to other Hsp90 inhibitors, studies revealed that only the Hsp90 C-terminus was capable of binding novobiocin. See Marcu et al., Novobiocin and Related Coumarins and Depletion of Heat Shock Protein 90-Dependent Signaling Proteins, J. Natl. Cancer Inst. 92, 242-248 (2000).

The function and location of the Hsp90 C-terminal domain is under intense investigation. Data gathered on the C-terminus is fairly indirect, as no Hsp90 co-crystal structure bound to C-terminal inhibitors has been reported. Unlike N-terminal inhibitors, it was found that occupation of the C-terminus by novobiocin results in displacement of ligands bound to the Hsp90 N-terminal ATP-binding site. See Marcu, M. G., Chadli, A., Bohouche, I., Catelli, B., Neckers, L. M. J. Biol. Chem., 276, 37181-37186 (2001); and Allan, R. K., Mok, D., Ward, B. K., Ratajczak, T. J. Biol. Chem., 281, 7161-7171 (2006). It has been proposed that novobiocin may induce a conformational change that results in disassembly of the Hsp90 homodimer and subsequent release of protein substrate. See Burlison, J. A., Blagg, B. S. J. Org. Lett., 8, 4855-4858 (2006). Compounds exhibiting higher affinity are likely to provide greater investigation of the Hsp90 C-terminal nucleotide-binding pocket and perhaps, afford a new class of Hsp90 modulators that could be used for the treatment of cancer.
The poor Hsp90 inhibitory activity of novobiocin has been improved through the preparation of various analogues of the coumarin ring and benzamide side chain by several groups. However, investigation of the sugar appendage of novobiocin has been limited and little is known about the structure-activity-relationship (“SAR”) of this moeity. Studies by the present inventor produced a library of compounds that established the first structure-activity relationships for novobiocin as an Hsp90 inhibitor. This focused library explored the features of the benzamide side chain, coumarin core and noviose sugar. These studies highlighted that both attachment of the noviose moiety to the 7-position and an amide linker at the 3-position of the coumarin ring are critical for anti-Hsp90 activity. Moreover, 2′ and 3′ modifications to the sugar identified the diol as most potent, over the carbonate and corresponding carbamates. See Yu et al., Hsp90 Inhibitors Identified from a Library of Novobiocin Analogues. J. Am. Chem. Soc., 127, 12778-12779 (2005).
N-(7-((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy)-2-oxo-2H-chromen-3-yl)acetamide (KU-1/A4) was identified the most notable compound identified from this library, having an IC50 of about 700 μM in SkBr3 cells. Compound A4, with a shortened N-acyl side chain, absent 4-hydroxy substituent and lack of carbamoyl group on the noviose sugar, induced degradation of Hsp90-dependent client proteins at about 70-fold lower concentration than novobiocin. Most importantly, compound A4 induced Hsp90 at concentrations 1000-10000-fold lower than that required for client protein degradation, a phenomenon that had not been observed with other Hsp90 inhibitors. Because of its activity and lack of toxicity, A4 was evaluated as a neuroprotective agent and was found to produce an EC50 at 6 nM, while exhibiting no toxicity at any concentration tested, in a model for Alzheimer's disease.
See Ser. No. 11/266,149 filed on Nov. 3, 2005, which is incorporated by reference.
To confirm the SAR trends observed, two natural product analogues, DHN1 and DHN2, were designed and subsequently evaluated. This study was aimed at specifically identifying essential functionalities of novobiocin responsible for its DNA gyrase inhibition. These novobiocin analogues confirmed that the 4-hydroxyl and the 3′-carbamate are detrimental to Hsp90 inhibitory activity, but critical for DNA gyrase inhibition. DHN1 and DHN2 exhibited an IC50 of about 7.5 and 0.5 μM in SkBr3 cells, respectively. See Burlison et al., Novobiocin. Redesigning DNA Gyrase Inhibitors for Selective Inhibition of Hsp90, J. Am. Chem. Soc., 128, 15529-15536 (2006). This study confirmed that the trends observed with the A4 library and produced the first selective inhibitors of the Hsp90 C-terminus.

SAR of the coumarin core of novobiocin has also been developed in a combinatorial library of coumarin analogues aimed at Hsp90 inhibition. See Huang, Y.-T., Blagg, B. S. J. J. Org. Chem., 72, 3609-3613 (2007).
Renoir and co-workers recently examined the role of noviose in Hsp90 inhibition. Their structure-activity relationship studies demonstrated that Hsp90 inhibition is observed in analogues that lack the noviose moiety, if a tosyl substituent is included at the C-4 or C-7 of the coumarin. These analogues manifested mid-micromolar IC50 values. See Le Bras, G., Radanyi, C., Peyrat, J.-F., Brio, J.-D., Alami, M., Marsaud, V., Stella, B., Renoir, J.-M. J. Med. Chem., 50, 6189-6200 (2007). In a subsequent paper, the same group suggested that Hsp90 inhibition can be enhanced by removal of C7/C8 substituents in denoviose analogues bearing a tosyl group at the 4-position. These studies produced inhibitors with simplified coumarins that also exhibited mid-micromolar IC50 values. See Radanyi, C., Le Bras, G., Messaoudi, S., Bouclier, C., Peyrat, J.-F. Brion, J.-D., Marsaud, V., Renoir, J.-M., Alami, M. Bioorg. Med. Chem. Lett., 18, 2495-2498 (2008).
While both the coumarin and benzamide side chain of the novobiocin scaffold have been probed, very limited SAR for the sugar portion exists. The present invention is directed to novel novobiocin analogues having a modified sugar.